Process for the preparation of 7-aminocephalosporanic acid

ABSTRACT

7-AMINOCEPHALOSPORANIC ACID, A VALUABLE INTERMEDIATE FOR THE PREPARATION OF SEMI-SYNTHETIC CEPHALOSPORINS, IS PREPARED BY A PROCESS COMPRISING THE CONSECUTIVE STEPS OF: (A) ACYLATING CEPHALOSPORIN C BROTH WITH AN ISOCYANATE TO PRODUCE A COMPOUND CALLED N-(N&#39;&#39;-ALKYL- OR ARYLCARBAMOYL)CEPHALOSPORIN C AND HAVING THE FORMULA   2-(HOOC-),3-(CH3-COO-CH2-),7-(HOOC-CH(-NH-CO-NH-R)-   (CH2)3-CO-NH-)-2-CEPHEM   IN WHICH R IS (LOWER)ALKYL OR ARLY; (B) RECOVERING DERIVATIVE III BY SOLVENT EXTRACTION; (C) SILYLATING THE CARBOXYL FUNCTIONS OF COMPOUND III TO FORM SILYL ESTERS; (D) HALOGENATING THE SILYL ESTER OF COMPOUND III TO PRODUCE AN IMINO-HALIDE; (E) FORMING AN INIMO-ETHER FROM THE IMINO-HALIDE BY TREATMENT WITH AN ALCOHOL; AND (F) MIXING SAID IMINO-ESTER WITH WATER OR AN ALCOHOL TO PRODUCE 7-AMINOCEPHALOSPORANIC ACID (7-ACA).

United States Patent ABSTRACT OF THE DISCLOSURE 7-aminocephalosporanicacid, a valuable intermediate for the preparation of semi-syntheticcephalosporins, is prepared by a process comprising the consecutivesteps of: (A) acylating cephalosporin C broth with an isocyanate toproduce a compound called N-(N-alkylor arylcarbamoyl)cephalosporin C andhaving the formula I II I S now-p-wmM-c-rz l 0 NH ll 1 O N CH2OCCHs I :0a 17111 0on1 R (III) in which R is (lower)alkyl or aryl;

(B) recovering derivative III by solvent extraction;

KC) silylating the carboxyl functions of compound III to form silylesters;

(D) halogenating the silyl ester of compound III to produce animino-halide;

(E) forming an imino-ether from the imino-halide by treatment with analcohol; and

(F) mixing said imino-ether with water or an alcohol to produce7-aminocephalosporanic acid (7-ACA).

BACKGROUND OF THE INVENTION (1) Field of the invention This inventionrelates to a novel process for the in situ preparation, subsequentharvesting and conversion of cephalosporin C derivatives into 7-ACA.

It is thus an object of the present invention to provide a new andimproved process for the preparation of 7-ACA.

(2) Description of the prior art Recovery methods for cephalosporin Cfrom fermentation broth are described in US. Pats. Nos. 3,093,638 and3,094,527 wherein the processes described involve adsorption of thecephalosporin C onto an adsorbant as compared to solvent extraction.While (lower) alkylcarbamoylor arylcarbamoyl-cephalosporin C derivativesare not described as being formed in situ in cephalosporin Cfermentation broth, or as being useful to aid in the extraction ofcephalosporin C from its broth, or as being useful as a startingmaterial in the production of 7-ACA, some of these compounds aredescribed in the patent literature (e.g. British Pat. 1,064,495 and US.Pat. 3,227,712) as antibiotics. Several processes for the chemicalcleavage of cephalosporin C or certain of its derivatives are describedin the patent literature (U.S. Pats. Nos. 3,188,311, 3,234,223,3,124,576 and British Pat. 1,041,985). None of these processes employ anN-(lower)alkylor arylcarbamoyl-cephalosporin C and a silyl ester thereofas a starting material.

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SUMMARY OF THE INVENTION This invention relates to and has for itsobject the provision of an improved process for the in situ preparationand recovery of a cephalosporin C derivative, said derivative beingutilizable in a chemical cleavage, as its silyl ester, to7-aminocephalosporanic acid.

7-aminocephalosporanic acid (7-ACA) is a most valuable intermediate inthe preparation of a multitude of semi-synthetic cephalosporanic acidantibacterial agents. In view of the impracticability of the totalsynthesis of 7-ACA on a large scale, commercial supplies of the compoundare prepared by the chemical degradation of naturally occurringcephalosporanic acids, i.e., cephalosporin C, which is produced byfermentation. Most 7-ACA is derived from cephalosporin C (U.S. Pat.3,093,638) which has the structure The production of 7-ACA by currentlyavailable methods is fraught with difficulties from the fermentation tothe chemical cleavage of cephalosporin C. Low yields of 7-ACA have madeit difficult for cephalosphoranic acids to take their rightful place inantibiotic therapy. For this reason the processes of the presentinvention are a significant improvement over the methods of the priorart.

Cephalosporin C is characterized by an amino-acid function in its sidechain. The amino-acid exists in the form of a zwitterion in aqueoussolution and as such is very water soluble. Because of its highly ionicnature, it is extremely difiicult to harvest by solvent extraction ofthe fermentation broth. The harvesting procedure currently used involvesthe adsorption of the crude cephalosporin C from the fermentation brothonto a suitable adsorbant, i.e., charcoal, an ion exchanger resin, orthe like, followed by elution, concentration and precipitation at theisoelectric point or by salt formation (U.S. Pat. No. 3,094,527). Thelow degree of efiiciency coupled with the complexity of each step inthis process combine to make it very difiicult to manufacture 7-ACA.

The building block of the semi-synthetic cephalosporins is 7-ACA, whosestructure is shown below:

HgN

l l O C 02H (II) Most 7-ACA is prepared by partial degradation ofcephalosporin C or its derivatives by chemical means (U.S. Pats. Nos.3,124,576, 3,188,311 and 3,234,223).

Again, most of these methods provide commercial yields that areundesirably low. Furthermore, these methods invariably employ as thestarting material a purified form of cephalosporin C Which is diflicultto obtain.

It was therefore an object of the present invention to improve on theharvestable yields of cephalosporin C from its fermentation broth insuch a chemical form as to be directly useable in subsequent cleavagereactions to produce 7-ACA Without substantial purification procedures.

The object of the present invention has been achieved by the provision,according to the present invention, of the process for the in situpreparation and harvesting of a derivative of cephalosporin C having theformula (III wherein R is (lower)alkyl but preferably ethyl, n-propyl,isopropyl, n-butyl or isobutyl; or a group of the formula wherein n isan integer of to 6 and R and R are alike or different and each is H, Cl,Br, F, N0 (lower)alkyl or (lower)alkoxy, but preferably hydrogen; whichcomprises the consecutive steps of:

(A) Adding an isocyanate having the formula in which R is as definedabove, to a previously filtered and acid-incubated fermentation brothcontaining cephalosporin C, in a ratio of at least 2 moles of isocyanateper mole of cephalosporin C, but preferably in a ratio of about 2 to 10moles of isocyanate per mole of cephalosporin C, and most preferably 5to 8 moles of isocyanate per mole of cephalosporin C, at a pH of about 7to 9, but preferably about 8, at a temperature in the range of about 20to 60 C., but preferably in the range of about -5 C. to about 20 C. toform said derivative of cephalosporin C; and

(B) Recovering said derivative of cephalosporin C preferably byextraction using a water-immiscible organic solvent such as methylisobutyl ketone, butanol, ethyl acetate or the like, but preferablybutanol, at a pH of about 1 to 3, but preferably about pH 2.

The term (lower)alkyl for the purpose of the present invention isdefined as an alkyl group comprised of 1 to 10 carbon atoms, includingfor example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, n-pentyl, etc., and the like, but especially methyl, ethyl,n-propyl, isopropyl, n-butyl and isobutyl. The terms (lower) alkoxy andhalo(lower)alkyl are also defined as moieties containing 1 to 10 carbonatoms.

The fermentation of the mold known as cephalosporium, or a mutatethereof, produces a mixture of compounds, predominately cephalosporin Cand lesser amounts of cephalosporin N (now known to be a penicillin).Cephalosporin C is acid stable while cephalosporin N is not.Accordingly, prior to the reaction of the fermentation broth with anisocyanate, it is desirable to destroy in situ any co-producedcephalosporin N. This is accomplished by acidification to about pH 2with a mineral acid such as hydrochloric acid, sulfuric acid, phosphoricacid, or the like, followed by an incubation period of about 2 to 20hours. The fermentation broth may be filtered before or afteracidification and incubation.

Following this step, the filtrate-fermentation broth containing thecephalosporin C is adjusted to about pH 7 to 9, but preferably'about 8,by the addition of an alkali metal base such as sodium or potassiumhydroxide.

The volume of the prepared broth is increased about 25% by the additionof acetone. The isocyanate is added to this solution with stirring in aratio of at least 2, preferably about 2 to 10 moles of isocyanate permole of cephalosporin C (broth concentration having been predetermined)but more preferably in a ratio of about 5 4 to 8 moles. The pH is heldconstant during the slow addition and for at least 30 minutes followingthe addition. The temperature of the broth is maintained below 40 C.,but preferably at 0 C. to 15 C. during this time. The reaction isusually complete one hour after the addition is completed.

One-half volume of an immiscible organic solvent is added, preferablyn-butanol, and the pH is adjusted to about 1 to 3, but preferably 2. Themixture is stirred and the organic solvent phase containing thecephalosporin C derivative III is collected.

The organic solution is concentrated in vacuo to about one-third itsoriginal volume at a temperature not to exceed 40 C. The concentrate iscooled to about 20 C. to 30 C. and the sodium salt of the cephalosporinderivative III is precipitated by the addition of a slight excess ofsodium 2-ethylhexanoate dissolved in n-butanol. The mixture is cooled to0 C. to 5 C. for 3 to 4 hours and the solid sodium salt of derivativeIII is collected by filtration. The filter cake is washed with coldbutanol followed by an n-hexane washing. The product is dried in vacuoat about 60 C.

It is another object of the present invention to provide a superiorprocess for the preparation of 7-ACA which is capable of using thecompounds having Formula III without the necessity of preliminarypurification or chemical conversion to cephalosporin C.

This object of the present invention has been achieved by the provisionaccording to the present invention, of the process for the preparationof 7-aminocephalosporanic acid which comprises the consecutive steps of:

(A) Mixing a compound having the formula MO 0 (3 (CH) (i l /S l I 0 NHGIN n orn-o-o-om f I IIIH 002M R (III) in which each M is selected fromthe group consisting of hydrogen, metal and amine cations, and R is(lower) alkyl, but preferably ethyl, n-propyl, isopropyl, n-butyl andisobutyl; or a group of the formula wherein n is an integer of 0 to 6and R and R are alike or different and each is H, Cl, Br, F, N0(lower)alkyl or (lower)alkoxy, but preferably hydrogen; with at leasttwo equivalents of a silyl compound of the formula f ins... R5 l R X orr /Si /R Ill 1 R NSi H Rs --N wherein R is hydrogen or (lower)alkyl andR is hydrogen, (lower)alkyl or but preferably dimethyldichlorosilane ortrimethylchlorosilane; under anhydrous conditions in the presence of anexcess of an acid deactivating tertiary amine selected from the groupconsisting of triethylamine, dimethylaniline, quinoline, lutidine,pyridine, or the like, in an inert solvent selected from the groupconsisting of methylene chloride, dichloroethane, chloroform,tetrachloroethane, nitromethane, diethylether, or the like, to producethe corresponding silyl di-ester of compound III;

(B) Mixing said silyl ester with a halogenating agent selected from thegroup consisting of phosphorous pentachloride, phosphorous pentabromide,phosphorous trichloride, phosphorous tribromide, oxalyl chloride,p-toluenesulfonyl halide, phosphorous oxychloride, phosgene, or thelike, in a molar ratio of 2 or more moles of halogenating agent per moleof silyl ester, but preferably about two moles, under anhydrousconditions in an inert solvent such as methylene chloride,dichloroethane, chloroform, tetrachloroethane, nitromethane, diethylether or the like, in the presence of an acid deactivating tertiaryamine selected from the group consisting of triethylamine,dimethylaniline, quinoline, lutidine, pyridine or the like, attemperatures below C., but preferably in the range of 40 C. to 60 C., toproduce in solution the corresponding imino-halide;

(C) Mixing with said solution of imino-halide an alcohol selected fromthe group consisting of aliphatic alcohols having 1 to 12 carbon atomsand phenylalkyl alcohols having 1 to 7 alkyl carbon atoms, at atemperature below 0 C., but preferably -40 C. to 70 C. to produce in thesolution the corresponding imino-ether; and

(D) Mixing said solution of imino-ether under acidic conditions withwater or an aliphatic alcohol, or a mixture of both, to produce7-aminocephalosporanic acid.

The process of the invention unexpectedly produces high yields underboth laboratory and commercial scale conditions. The yields, which maybe in the order of 50% to 70%, are attributed to the use of silyl esterson the carboxyl groups of the compounds of Formula III. The silyl estersmay be prepared and hydrolyzed to the acid again Without the loss ofproduct, especially if the reaction proceeds at the temperatures belowC., preferably -40 C. to -70 C., during formation of the imino-ether.Moreover, the use of silyl esters rather than the esters employed in thepreviously cited patents simplifies the process since the silyl esterhydrolyzes simultaneously with the splitting of the double bond of theimino-group and avoids the separate step of splitting the 4 carboxylicesters of the prior art processes.

The formation of the silyl ester is accomplished by reacting a silylcompound of Formula IV or V, under anhydrous conditions, in an inertorganic solvent, with the compound of Formula III, or a salt thereof, inthe presence of an acid deactivating tertiary amine.

Suitable inert solvents include amongst others methylene chloride,dichloromethane, chloroform, tetrachloroethane, nitromethane, benzeneand diethyl ether.

Suitable salts of compound III include amongst others alkali metal andalkaline earth metal salts such as potassium, sodium, calcium, aluminumetc. Also acceptable are ammonium and amine salts, preferably tertiaryamines such as triethylamine, dibenzylamine, trimethylamine,N-methylmorpholine, pyridine, N benzyl B phenethylamine, 1 ephenamine,N,N' dibenzylethylene diamine, dehydroabietylamine,N-(lower)-alkylpiperidines such as N-ethylpiperidine, and the like.Tertiary-amine salts are preferable.

Suitable acid deactivating tertiary amines include amongst otherstriethylamine, dimethylaniline, quinoline, lutidine, pyridine, etc. Thequantity of acid deactivating amine used is preferably an amountequivalent to about 75% of the total acid generated in the process bythe halogenating agent and the halosilane compound reacting withcompound III.

Suitable silyl compounds of Formulas IV and V are: trimethylchlorosilane, hexamethyl disilazane, triethyl chlorosilane, methyltrichlorosilane, dimethyl dichlorosilane, triethyl bromosilane,tri-n-propyl chlorosilane, bromomethyl dimethyl chlorosilane,tri-n-butyl chlorosilane, methyl diethyl chlorosilane, dimethyl ethylchlorosilane, phenyl dimethyl bromosilane, benzyl methyl ethylchlorosilane, phenyl ethyl methyl chlorosilane, triphenyl chlorosilane,triphenyl fluorosilane, tri-o-tolyl chlorosilane,tri-p-dimethylaminophenyl chlorosilane, N-ethyl triethyl silylamine,hexaethyl disilazane, triphenyl silylamine, tri-n-propyl silylamine,tetraethyl dimethyl disilazane, etc. The same effect is produced byhexa-alkyl phenyl disilazane, hexaphenyl disilazane, hexa-p-tolyldisilazane, etc. The same effect is produced byhexaalkylcyclotrisilazanes or oeta-alkylcyclotetrasilazanes. Othersuitable silylating agents are silylamides and silylureides such astrialkylsilylacetamide and a bis-trialkylsilylacetamide.

The imino compound is preferably an imino chloride or bromide which canbe prepared by reacting the silyl ester of compound III with ahalogenating agent such as phosphorus pentachloride, phosphoruspentabromide, phosphorus trichloride, phosphorus tribromide, oxalylchloride, p-toluene sulfonic acid halide, phosphorus oxychloride,phosgene, etc., under anhydrous conditions in the presence of acidbinding agents at temperatures preferably below 0 C. such as 40 to 60 C.

A very important step for high yields of the process of the presentinvention is the formation of the imino ether by reacting the iminohalide under anhydrous conditions with a primary or secondary alcohol attemperatures between 20 and 70 C., preferably about 40 C. to 70 C.Temperatures higher than -40 C. result in a substantial reduction inyield.

Suitable alcohols for producing the imino ethers from the imino-halidesare primary and secondary alcohols having the general formula R OH inwhich R is selected from the group consisting of (A) (lower)alkyl,having 1 to 12 carbon atoms, preferably having 1-3 carbon atoms, such asmethanol, ethanol, propanol, isopropanol, n-butanol, amyl alcohol,decanol, etc.; (B) phenylalkyl having 1 to 7 alkyl atoms, such as benzylalcohol, 2- phenylethanol-l, etc.; (C) cycloal-kyl ,such ascyclohexylalcohol, etc; (D) hydroxyalkyl having 2 to 12 carbon atoms,preferably at least 3 carbon atoms, such as 1,6- hexanediol, etc.; (E)alkoxyalkyl having 3 to 12 carbon atoms, such as 2-methoxyethanol,2-isopropoxyethanol, 2-butoxyethanol, etc.; (F) aryloxyalkyl, having 3to 7 carbon atoms in the aliphatic chain, such as2-p-chlorophenoxyethanol, etc.; (G) aralkoxyalkyl, having 3 to 7 carbonatoms in the aliphatic chain, such as 2-(p-methoxybenzyloxy)-ethanol,etc.; (H) hydroxyalkoxyalkyl, having 4 to 7 carbon atoms, such diglycol.Also mixtures of these alcohols are suitable for forming the iminoethers.

After formation of the imino-ether from the iminohalide, the imino bondmust be split to produce 7-ACA. The process is accomplished by mildhydrolysis or alcoholysis. If the quantity of acid deactivating tertiaryamine present in the process is a quantity less than the acid producedby the silylation and halogenation, the cleavage will probably proceedsimultaneously upon completion of the formation of the imino-ether. Ifhowever the quantity of acid deactivating amine was more than the acidproduced, the cleavage step will require the careful addition of aquantity of H+ to effect the cleavage.

The 7-ACA is harvested from the reaction mixture by adjusting the pH ofthe mixtures to or near the isoelectric point of the 7-ACA, followingwhich the 7-ACA crystallizes and is collected by filtration.

For optimum yields of 7-ACA, it is preferred to use high concentrationsof the reactants. For example, in the formation of the silyl esters, a20% to 30%, preferably 25% by Weight of compound III is suspended in aninert organic solvent and a base for the best results. The silane isemployed preferably in excess, i.e. 10% to 60%, above theoreticalamounts.

One molecule of cephalosporin C derivative HI possesses two reactivecarboxyl groups capable of forming silyl esters. Therefore, in terms ofthe silylation reaction, one mole of III is equal to two equivalentweights.

Accordingly, when compound III is treated with dichlorodimethylsilane,one molecule of compound III (two equivalent weights) is treated with atleast one molecule (two equivalent weights) of dichlorodimethylsilane.Similarly, when compound III is treated with chlorotrimethylsilane, onemolecule of compound I II (two equivalent weights) is treated with atleast .two molecules (two equivalent weights) of chlorotrimethylsilane.

This enables the use of solvents which are not absolutely dry becausetrace amounts of water are removed therefrom by reacting with the excesssilylating agent. Obviously the quantity of the silane compound requiredis dependent upon whether one or both carboxyl groups of the compoundIII are available for silyl ester formation. The reaction scheme isillustrated below:

8 DESCRIPTION OF THE PREFERRED EMBODIMENTS Example l.--In situpreparation of N-(N'-butylcarbamoyl)cephalosporin C Twenty-nine hundredliters of whole fermentation broth containing cephalosporin C was mixedwith 108 kg. of a filtering aid and 300 ml. of silicone antifoam' andthen filtered at pH 6.9 at 10 C.

Suflicient oxalic acid was added to the filtered broth to make the pH3.1. Following this addition, the pH was adjusted to pH 2 by theaddition of 30% sulfuric acid. Fresh filtering aid, 54 kg., was addedand the mixture was filtered. The tfiltrate thus obtained was extractedwith /2 volume of methyl isobutyl ketone (MIBK) at pH 2 and thenseparated. The MIBK phase was discarded.

One-fourth volume of acetone was added to the extracted broth and the pHwas adjusted to 7.85 with a solution of sodium hydroxide.n-Butylisocyanate (2"kg./ 1000 l. of broth) was added slowly withstirring (a molar The following examples will serve to illustrate butnot to limit the scope of the present invention.

ratio of about 6.1 moles of n-butylisocyanate per mole of cephalosporinC).

The pH was constantly maintained at pH 7.8 to 8.0 by the addition of 15%sodium hydroxide and the temperature was held in the range of C. to C.during the addition. Stirring was continued until the pH remainedconstant without the addition of further sodium hydroxide, a time ofabout 2 hours.

The pH was adjusted to pH 2 by the addition of 30% sulfuric acid and theacylated broth was extracted with three-fourths volume of n-butanol. Thebutanol phase was water-washed and then concentrated to approximatelyone-half the starting volume at 36 C. in vacuo. The water content of theconcentrate was negligible.

The concentrate was cooled and a solution of sodium ethylhexanoate inn-butanol was added to pH 4.8. The sodium salt ofN-(N-butylcarbamoyl)cephalosporin C precipitated and was collected byfiltration. The precipitate was washed with butanol, re-slurried inpetroleum ether, washed with acetone, filtered and dried in a vacuumoven at 40 C. to 60 C. to yield the product. Assay of the spent brothindicated the presence of less than cephalosporin C activity. Theproduct was of adequate purity for the subsequent preparation of 7-ACA.It was analyzed as the diacid.

Analysis.--Calcd for C H N O S-H O (percent): C, 47.36; H, 6.06; N,10.54; H O, 3.38. Found (percent): C, 47.85; H, 6.52; N, 10.54; S, 6.45;H O, 3.48.

Example 2.-Preparation of 7-aminocephalosporanic acid SodiumN-(N-butylcarbamoyl)cephalosporin C [13.4 grams], 45 milliliters ofmethylene chloride, 1.0 ml. of dimethylaniline and 3.67 ml. oftriethylamine were mixed together. Dichlorodimethylsilane (5.0 ml.) wasadded with stirring at a temperature of 28 C. The solution was stirredminutes. The solution was then cooled to 60 C. and 11.5 grams ofphosphorus pentachloride dissolved in 100 ml. of methylene chloride wasadded while the temperature of the reaction was kept below C. Anadditional 12.4 ml. of N,N-dimethylaniline in 10 ml. methylene chloridewas added. The temperature was held below 40 C. for 2 hours, thenchilled to 73 C., and a mixture of 60 ml. of methanol and 2.5 ml.dimethylaniline chilled to --78 C. was added slowly. The temperaturerose to 47 C. The mixture was stirred and recooled to '50 C. At the endof two hours, 55 ml. of water heated to +95 C. was added. Thetemperature rose to +5 C. The mixture was cooled in an ice bath andstirred for 4 minutes. The pH was 01. Ammonium hydroxide (21.5 ml.) wasadded over 8 minutes to a pH of 3.8. The mixture was stirred severalhours and then filtered. The precipitate was collected, washed with 25ml. of methylene chloride, then 25 ml. of water, then 50 ml. of methanoland finally 50 ml. of' acetone. The solid was dried to yield 4.05 gramsof 93.7% 7-ACA.

Example 3.-In situ preparation of sodium N-(N-phenylcarbamoyl)cephalosporin C Substitution in the procedure of Example1 for the 6.111 molar ratio of n-butylisocyanate:cephalosporin C usedtherein of a 7:1 molar ratio of phenylisocyanate produced a satisfactoryproduct for cleavage to 7-ACA. Assay of the spent broth after extractionindicated the presence of less than 15% cephalosporin C activity. It wasassayed as the diacid.

Analysis.-Calcd for C H N O S (percent): C, 51.68; H, 4.90; N, 10.48; S,6.00. Found (percent): C, 51.63; H, 5.27; N, 10.62; S, 6.14.

Example 4.Preparation of 7-aminocephalosporanic acid SodiumN-(N-phenylcarbamoyl)cephalosporin C [13.9 g.], milliliters of methylenechloride, 1 ml. of dimethylaniline and 3.67 ml. of triethylamine weremixed together. Dichlorodimethylsilane (5.5 ml.) was added with stirringat a temperature of 25 28 C. The slurry was stirred for 75 minutes, thencooled to -60 C. and 12.0 grams of PCl in 100 ml. of methylene chloridewas added. An additional 11.0 ml. of dimethylaniline in 10 ml. ofmethylene chloride was added. The temperature was maintained at 40 C.for 2 hours. The solution was cooled to -75 C. and a solution of 60 ml.methanol and 2.5 ml. dimethylaniline cooled to 78 C. was added. Thetemperature rose to 52 C. The temperature was held in the range of 45 C.to -50 C. for 2 hours. Fifty ml. of water warmed to C. was added withstirring. The temperature rose to +5 C. After 4 minutes of stirring (pH0.0), 22 ml. of NH OH was added over an 8 minute interval to pH 3.7. Theslurry was stirred for several days with cooling. The precipitated 7-ACAwas collected by filtration, washed with 50 ml. methylene chloride, 40ml. water, 50 ml. methanol, then 50 ml. acetone.

Example 5.In situ preparation of sodium N-(N-benzylcarbamoyl)cephalosporin C Substitution in the procedure of Example1 for the 61:1 molar ratio of n-butylisocyanate:cephalosporin C usedtherein of a 7.011 molar ratio of benzylisocyanate per mole ofcephalosporin C produces sodium N-(N benzylcarbamoyl) cephalosporin C.

Example 6.7-ACA from sodium N-(N'-benzylcarbamoyl)cephalosporin CSubstitution in the procedure of Example 2 for the sodium N (Nbutylcarbamoyl)cephalosporin C used therein of sodiumN-(N'-benzylcarbamoyl)cephalosporin C produces 7-ACA.

Example 7.Sodium N-(N-((lower))alkyl-or arylcarbamoyl)cephalosporin Cderivatives Substitution in the procedure of Example 1 for then-butylisocyanate used therein of and Example 8 Substitution in theprocedure of Example 2 for the sodium N (N' butylcarbamoyl)cephalosporinC used therein of the carbamoylcephalosporin C derivatives prepared inExample 7 produces 7-ACA.

While in the foregoing specification various embodiments of thisinvention have been set forth in specific detail and elaborated for thepurpose of illustration, it will be apparent to those skilled in the artthat this invention is susceptible to other embodiments and that many ofthe details can be varied widely without departing from the basicconcept and the spirit and scope of the invention.

1 1 We claim: 1. The process for the in situ preparation and harvestingof a carbamoyl derivative of cephalosporin C having the formula whereinR is (lower)alkyl or a group of the formula wherein n is an integer ofto 6 and R and R are alike or different and each is H, Cl, Br, F, N0(lower)alkyl or (lower) alkoxy; which comprises the consecutive stepsof:

(A) adding an isocyanate having the formula in which R is as definedabove, to an acid-incubated fermentation broth containing cephalosporinC, which was previously prepared by fermentation of a mold of thecephalosporium genus, in a ratio of at least 2 moles of isocyanate permole of cephalosporin C, at a pH above 7, at a temperature below 40 C.,to form said carbamoyl derivative of cephalosporin C; and (B) recoveringsaid carbamoyl derivative of cephalosporin C from the fermentation brothby extraction with a water-immiscible solvent. 2. The process of claim 1for the in situ preparation and harvesting of a carbamoyl derivative ofcephalosporin C having the formula wherein R is (lower)alkyl or a groupof the formula wherein n is an integer of 0 to 6 and R and R are alikeor different and each is H, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy;which comprises the consecutive steps of:

'(A) adding an isocyanate having the formula in which R is as definedabove to a previously filtered and acid-incubated fermentation brothcontaining cephalosporin C, which was previously prepared byfermentation of a mold of the cephalosporium genus, in a ratio of about2 to moles of isocyanate per mole of cephalosporin C, at a pH of about 7to 9, at a temperature in the range of about 0 C. to about 25 C. to formsaid carbamoyl derivative of cephalosporin C; and

(B) recovering said carbamoyl derivative of cephalosporin C byextraction using a water-immiscible organic solvent at a pH in the rangeof about 1 to 3.

r 12 3. The process of claim 1 for the in situ" preparation andharvesting of a carbamoyl derivative of cephalosporin C having theformula wherein R is (lower)alkyl or a group of the formula wherein n isan integer of 0 to 6 and R is H, Cl, Br, F, N0 (lower) alkyl or (lower)alkoxy; which comprises the consecutive steps of:

(A) adding an isocyanate having the formula in which R is as definedabove, to a previously filtered and acid-incubated fermentation brothcontaining cephalosporin C, which was previously prepared byfermentation of a mold of the cephalosporium genus, in a ratio of about5 to 8 moles of isocyanate per mole of cephalosporin C, at a pH of about7 to 9, at a temperature of about 0 C. to 15 C., to form said carbamoylderivative of cephalosporin C; and (B) recovering said carbamoylderivative of cephalosporin C by extraction using a water-immiscibleorganic solvent selected from the group consisting of methyl isobutylketone, ethylacetate, butanol, chloroform, methylene chloride anddichloroethane, at a pH in the range of about 1 to 3. 4. The process ofclaim 1 for the in situ preparation and harvesting of a derivative ofcephalosporin C having the formula wherein R is ethyl, n-propyl,isopropyl, n-butyl or isobutyl; or a group of the formula wherein n isan integr of 0 to 6; which comprises the consecutive steps of:

(A) adding an isocyanate having the formula in which R is as definedabove, to a previously fil tered and acid-incubated fermentation brothcontaining cephalosporin C,, which was previously prepared byfermentation of a mold of the cephalosporium genus, in a ratio of about6 moles of isocyanate per mole of cephalosporin C, at a pH of about 8,at a temperature of about 0 C. to about 5 C., to form said carbamoylderivative of cephalosprin C; and

(B) recovering said carbamoyl derivative of cephalosporin C byextraction using n-butanol, at a pH of about 2.

3,573,295 13 14 5. The process for the preparation of7-aminocephalotetrachloroethane, nitromethane or diethyl ether; insporanic acid which comprises the consecutive steps of: the presence ofan acid deactivating tertiary amine (A) mixing a compound having theformula such as trimethylamine, triethylamine, dimethylaniline,quinoline, lutidine or pyridine; at temperatures in the range of ---10C. to 60 C.; to produce in H o H I II I S solution the correspondingimino-halide; MO2C (CH2) C N I 0 (C) mixing With said solution ofimino-halide an al- IIH N OH 0 a CH cohol selected from the groupconsisting of aliphatic 0:0 V alcohols having 1 to 12 carbon atoms andphenyll 1 M 10 alkyl alcohols having 1 to 7 alkyl carbon atoms; at atemperature in the range of 20 C. to 70 C.; R to produce in the solutionthe corresponding iminoether; and

(D) mixing said solution of imino-ether under acidic conditions withwater or an aliphatic alcohol or a mixture of both; at a temperatureabout 0 C.; to produce 7-aminocephalosporanic acid.

in which each M is selected from the group consisting of hydrogen, metaland amine cations, and R is (lower)alkyl or a group of the formula R2 6.The process of claim 5 for the preparation of 7- aminocephalosporanicacid which comprises the consecutive steps of:

(A) mixing a compound having the formula wherein n is an integer of O to6 and R and R are alike or different and each is H, Cl, Br. F, N0 f ii f/S (lower)alkyl or (lower)alkoxy; 2 2)3 "ON:l I O with a silyl compoundof the formula III}? 0 N CH2-O H 5 Y R4 /R4" 17H 002M SiNH R5 R (III) RNH l or RS1X in which each M is selected from the group consist- 1 ingof hydrogen, metal and amine cations, and R is ethyl, n-propyl,isopropyl, n-butyl or isobutyl; or a u R m 39 group of the formulawherein R R and R are selected from the group consisting of hydrogen,halogen, (lower)alkyl, halo(lower) alkyl and phenyl, vat least one ofthe said R R and R' groups being other than halogen or hydrogen; R is(lower)alkyl, m is an integer of 1 to 2 and X is selected from the groupconsisting of halogen and wherein n is an integer of 0 to 6 and R is H,Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy; with a silyl compound ofthe formula R5 R R -N Si NH R5 4 R4 or Rfl S!iX wherein R is hydrogen or(lower)alkyl and R is a hydrogen, (lower)alkyl or R4 In R5 R5li whereinR R and R are selected from the group 1 consisting of hydrogen, halogen,(lower)alkyl, halo- (lower)alkyl and phenyl, at least one of the said Runder anhydrous conditions; in a ratio of at least one R and R groupsbeing other than halogen or hyequivalent or silylating agent perequivalent of comdrogen; R is (lower)alkyl, m is an integer of 1 to 2pound III; in the presence of an acid deactivating terand X is selectedfrom the group consisting of halotiary amine selected from the groupconsisting of trigen and ethylamine, trimethylamine, dimethylaniline,quinoline, lutidine and pyridine; in an inert solvent selected from thegroup consisting of methylene chloride, dichloroethane, chloroform,tetrachloroethane, R nitro-methane and diethyl ether; to produce thecorresponding silyl ester of compound III;

(B) mixing said silyl ester with a halogenating agent selected from thegroup consisting of phosphorus pen- Wherein R is hydrogen or(lower)alkyl and R is hydrogen, (lower)alkyl or tachloride, phosporuspentabromide, phosphorus trichloride, phosphorus tribromide, oxalylchloride, p- R Si toluenesulfonyl halide, phosphorus oxychloride andphosgene; in a molar ratio of 2 to 4 mole of halogenating agent per moleof silyl ester; under anunder anhydrous conditions in a ratio of about1.2 hydrous conditions; in an inert organic solvent such to about 2equivalents of silyating agent per equivas methylene chloride,dichloroethane, chloroform, alent of compound III, in the presence of anacid deactivating tertiary amine selected from the group consisting oftrimethylamine, triethylamine, dimethylaniline, quinoline, lutidine andpyridine in an inert organic solvent selected from the group consistingof methylene chloride, dichloromethane, chloroform, tetrachloroethane,nitromethane and diethyl ether, to produce the corresponding silyl esterof compound III;

(B) mixing said silyl ester with a halogenating agent selected from thegroup consisting of phosphorus pentachloride and phosphorus oxychloride,in a molar ratio of 2 to 3 moles of halogenating agent per mole of silylester, under anhydrous conditions in methylene chloride, dichloroethane,chloroform or tetrachloroethane, in the presence of a tertiary aminesuch as triethylamine, dimethylaniline or pyridine, at temperatures inthe range of 40 C. to -60 C., to produce in solution the correspondingirrnino-halide;

(C) mixing with said solution of imino-halide methanol, ethanol,n-propanol or isopropanol, at a temperature in the range of 40 C. to 70C., to produce in the solution the corresponding iminoether; and

(D) mixing said solution of imino-ether under acidic conditions withwater or an alcohol selected from the group consisting of methanol,ethanol, n-propanol or isopropanol, or a mixture thereof, at atemperature in the range of about 10 C. to about +10 C., to produce7-aminocephalosporanic acid.

7. The process of claim 6 wherein R is ethyl, n-propyl, isopropyl,n-butyl, isobutyl, phenyl or phenethyl; the silyl compound isdimethyldichlorosilane or trimethylchlorosilane; the inert organicsolvent is methylene chloride or dichloroethane; and the halogenatingagent is phosphorus pentachloride and it is used in a molar ratio of twomoles of phosphorus pentachloride per mole of silyl ester.

8.. The process for the in situ preparation and harvesting of aderivative of cephalosporin C and its subsequent cleavage to7-aminocephalosporanic acid which comprises the consecutive steps of:

(A) adding to a previously filtered and acid incubated fermentationbroth containing cephalosporin C 'which was previously prepared byfermentation of a mold of the cephalosporium genus an isocyanate havingthe formula wherein R is (lower)alkyl or a group of the formula whereinn is an integer of to 6 and R and R are alike or difierent and each isH, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy; in a ratio of about 2 to10 moles of isocyanate per mole of cephalosporin C; at a pH of about 7to '9; at a temperature in the range of about 0 C. to about 25 C.; toproin which R is as defined above;

(B) recovering said derivative of cephalosporin C by extraction using awater-immiscible organic solvent at a pH in the range of about 1 to 3;

(C) mixing the cephalosporin C derivative, or a salt thereof, with asilyl compound of the formula wherein R R and R are selected from thegroup consisting of hydrogen, halogen, (lower) alkyl, halo- (lower)alkyland phenyl, at least one of the said R R and R groups being other thanhalogen or hydrogen; R is (lower)alkyl, m is an integer of 1 to 2 and Xis selected from the group consisting of halogen and wherein R ishydrogen or (lower) alkyl and R is hydrogen, (lower)alkyl or R5 R 's iunder anhydrous conditions, in a ratio of at least one equivalent ofsilylating agent per equivalent of compound III, in the presence of anacid deactivating tertiary amine selected from the group consisting oftriethylamine, trimethylamine, dimethylaniline, quinoline, lutidine andpyridine, in an inert solvent selected from the group consisting ofmethylene chloride, dichloroethane, chloroform, tetrachloroethane,nitromethane and diethyl ether, to produce the corresponding silyl esterof compound HI;

(D) mixing said silyl ester with a halogenating agent selected from thegroup consisting of phosphorus pentachloride, phosphorus pentabromide,phosphorus trichloride, phosphor-us tribromide, oxalyl chloride,p-toluenesulfonyl halide, phosphorus oxychloride and phosgene, in amolar ratio of 2 to 4 moles of halogenating per mole of silyl ester,under anhydrous conditions in methylene chloride, dichloroethane,chloroform, tetrachloroethane, nitromethane, in the presence oftrimethylamine, triethylamine, dimethylaniline, quinoline, lutidine orpyridine, at tempera tures in the range of -10 C. to -60 C. to producein solution the corresponding imino-halide;

(E) mixing with said solution of imino-halide an alcohol selected fromthe group consisting of aliphatic alcohols having 1 to 12 carbon atomsand phenyl alkyl alcohols having 1 to 7 alkyl carbon atoms, at atemperature in the range of 20 C. to C. to produce in the solution thecorresponding iminoether; and

(F) mixing said solution of imino-ether under acidic conditions withwater or an aliphatic alcohol, or a mixture of both, at a temperatureabout 0 C., to produce 7-arninocephalosporanic acid.

9. The process of claim 8 which comprises the consecutive steps of:

(A) adding to a previously filtered and acid-incubated fermentationbroth containing cephalosporin C which was previously prepared byfermentation of a mold of the cephalosporium genus an isocyanate havingthe fonrnula wherein R is n-propyl, n-butyl, isobutyl, or phenyl; 1n aratio of about 6 moles of isocyanate per mole of cephalosporin C; at apH of about 8; at a tempera- 1 7 1 8 ture of about C. to about C.; toproduce a (D) mixing said silyl ester with phosphoruspentachloderivative of cephalosporin C having the formula ride, in amolar ratio of,2 to 3 moles of halogenating agent per mole of silylester, under anhydrous conditions in methylene chloride, chloroform, ordichloro- S ethane, in the presence of triethylamine, dimethyl- 5aniline or pyridine, at temperatures in the range of llIH H C. to C., toproduce in solution the cor- CH2 OT responding imino-halide; I l (E)mixing with said solution of imino-halide, meth- I COZH l0 anol,ethanol, n-propanol or isopropanol, at a tem- R 1 perature in the rangeof 40 C. to C. to

- produce in the solution the corresponding iminoin which R is asdefined above; ether} f (B) recovering said derivative of cephalosporinC by (F) salfi solutlon 0f lmlno'ether under acldic extraction usingmbutanol at a PH of about 15 conditions with Water or methanol, ethanol,n-pro- (C) mixing the cephalosporin C derivative, or a salt Pan01 OI{sopropanol or a mlxturfi thereof, at a thereof, withdimethyldichlorosilane or trimethylchlo- Pmamre m the range of aboutabout rosilane under anhydrous conditions, in a ratio of to Produce7'amlnocephalosporanlc acidabout 1.2 to 2 equivalents of silylatingcompound per equivalent of compound III, in the presence of 20References Cited an acid deactivating group selected from the groupUNITED STATES PATENTS consisting of trimethylamine, triethylamine,dimeth- 3,499 909 3/1970 Weissenburger et a1. 7 ylaniline, quinoline,lutidine and pyridine, in methyl- 3993:6323 6/1963 Abraham et aL 260243'C ene chloride or dichloroethane, to produce the corresponding silylester of compound III; 25 NICHOLAS S. RIZZO, Primary Examiner

